Adenovirus vector carrying REIC/DKK-3 gene: neoadjuvant intraprostatic injection for high-risk localized prostate cancer undergoing radical prostatectomy.

As the First-In-Human study of in situ gene therapy using an adenovirus vector carrying the human REIC (reduced expression in immortalized cell)/Dkk-3 gene (Ad-REIC), we conducted neoadjuvant intraprostatic injections in patients with high-risk localized prostate cancer undergoing radical prostatectomy (RP). Patients with recurrence probability of 35% or more within 5 years following RP, as calculated by Kattan's nomogram, were enrolled. Patients received two ultrasound-guided intratumoral injections at 2-week intervals, followed by RP 6 weeks after the second injection. After confirming the safety of the therapeutic interventions with initially planned three escalating doses of 1.0 × 1010, 1.0 × 1011 and 1.0 × 1012 viral particles (vp) in 1.0-1.2 ml (n=3, 3 and 6), an additional higher dose of 3.0 × 1012 vp in 3.6 ml (n=6) was further studied. All four DLs including the additional dose level-4 (DL-4) were feasible with no adverse events, except for grade 1 or 2 transient fever. Laboratory toxicities were grade 1 or 2 elevated aspartate transaminase/alanine transaminase (n=4). Regarding antitumor activities, cytopathic effects (tumor degeneration with cytolysis and pyknosis) and remarkable tumor-infiltrating lymphocytes in the targeted tumor areas were detected in a clear dose-dependent manner. Consequently, biochemical recurrence-free survival in DL-4 was significantly more favorable than in patient groups DL-1+2+3.

Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer.

Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

The induction of antigen-specific CTL by in situ Ad-REIC gene therapy.

An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

Integrin antagonist augments the therapeutic effect of adenovirus-mediated REIC/Dkk-3 gene therapy for malignant glioma.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) was identified as a gene whose expression is reduced in many human cancers. REIC/Dkk-3 expression is also downregulated in malignant glioma and regulates cell growth through caspase-dependent apoptosis. cRGD (EMD121974), an antagonist of integrins, has demonstrated preclinical efficacy against malignant glioma. In this study, we investigated the antiglioma effect of combination therapy using an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) and cRGD. Quantitative real-time reverse-transcription PCR revealed the reduction of REIC/Dkk-3 mRNA levels in malignant glioma cell lines. The reduction of REIC/Dkk-3 protein expression in malignant glioma cell lines was also confirmed with western blot analysis. After treatment with Ad-REIC and cRGD, the proliferative rate of malignant glioma cells was significantly reduced in a time-dependent manner. In vivo, there was a statistically significant increase in the survival of mice treated with Ad-REIC and cRGD combination therapy compared with Ad-REIC monotherapy. We identified an apoptotic effect following monotherapy with Ad-REIC. Moreover, cRGD augmented the antiglioma efficacy of Ad-REIC. These results may lead to a promising new approach for the treatment of malignant glioma.

Potent antitumor effects of combined therapy with a telomerase-specific, replication-competent adenovirus (OBP-301) and IL-2 in a mouse model of renal cell carcinoma.

OBP-301 (a telomerase-specific, replication-competent adenovirus with hTERT promoter) was constructed in a previous study and it showed a strong anticancer effect by inducing cell lysis in human lung and prostate cancer cells. This study investigated the effectiveness of a combination therapy of OBP-301 and interleukin-2 (IL-2) in a mouse model of renal cell carcinoma (RCC). The cell-killing effect of OBP-301 was confirmed in vitro in the RENCA cancer cells. In in vivo experiment, luciferase-expressing RENCA cells were implanted in the left kidney and lung of BALB/c mice to prepare the RCC metastatic model. The animals were randomly divided into four treatment groups: PBS, IL-2 alone, OBP-301 alone and the combination. The analyses of orthotopic tumor weight, lung metastasis and luciferin-stained tumor images 14 days after each treatment showed significant tumor growth inhibition in the combination group in comparison with that in the OBP-301- or IL-2-treated groups. In addition, the percentage of regulatory T-cells (Tregs) in the combination group was significantly suppressed in comparison with that in the PBS and single-agent treatment groups. The outcomes of this study suggest that tumor-specific oncolytic immunovirotherapy may become an attractive strategy for the treatment of human RCC.

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer.

BACKGROUND: In light of the poor prognosis for cervical cancer, research continues into the development of innovative and efficacious treatment modalities for this disease. We investigated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) and evaluated its clinical importance in cervical cancer. PATIENTS AND METHODS: HAI-2 expression was examined in cervical cancer specimens (n=52) by immunohistochemistry. We further attempted to investigate the biological functions and inhibitory effects of HAI-2 using human papillomavirus (HPV) 16 type SiHa and HPV 18 type HeLa cervical cancer cell lines. RESULTS: There were significant correlations between HAI-2 expression and stage (P=0.017), lymph node metastasis (P=0.005) and ovarian metastasis (P=0.038). Low HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-2 expression (disease-free survival rate, P=0.016; overall survival rate, P=0.021). After transient transfection into the SiHa and HeLa cell lines, HAI-2 showed potential inhibitory effects mediated by reductions in hepsin and matriptase expression, which led to apoptosis by increasing the level of Bak and reducing the level of Bcl-2. CONCLUSIONS: The present findings indicate that low HAI-2 expression in cervical cancer may be associated with a poor prognosis. We propose that HAI-2 may represent a therapeutic target for the treatment of cervical cancer.

REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer.

The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH(2)-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.

Direct and distant antitumor effects of a telomerase-selective oncolytic adenoviral agent, OBP-301, in a mouse prostate cancer model.

We previously constructed OBP-301 (Telomelysin, a telomerase-specific replication-competent adenovirus with human telomerase reverse transcriptase (hTERT) promoter), which showed a strong anticancer effect by inducing cell lysis of human non-small cell lung cancer and colorectal cancer cells. To investigate the utility of OBP-301 for prostate cancer treatment, we herein evaluate the cell killing and antitumor effects. First, in vitro hTERT-specific adenovirus transduction in human prostate cancer cells (LNCaP, PC3, DU145) was confirmed using OBP-401 (Telomelysin-green fluorescent protein (GFP)). There was no detectable GFP transduction in the human prostate normal cells (PrEC, PrSC). Consistently, the cell-killing effect of OBP-301 was observed only in the cancer cells. Second, using an in vivo subcutaneous LNCaP tumor model in nude mice, we demonstrated that three intratumoral OBP-301 injections (10(7) PFU per tumor x 3 days) were sufficient to eradicate the detectable LNCaP prostate tumor. We also demonstrated that the ispilateral treatment with OBP-301 significantly suppressed contralateral LNCaP tumor growth in both sides of the tumor model. Histological and immunohistochemical analyses revealed diffuse oncolytic degeneration and adenoviral E1A protein expression in both sides of the tumors. Therefore, in situ OBP-301 administration could be a promising therapeutic strategy against prostate cancer and its metastatic lesions.

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Post-operative infection by pathogenic micro-organisms in the oral cavity of patients with prostatic carcinoma.

The aim of this study was to analyse the change in the oral cavity microflora of 14 patients who had undergone a radical prostatectomy for prostatic carcinoma. The detection of micro-organisms in the oral cavity was compared before and after the surgical procedure. Post-operative infection, defined as those patients who had increased Candida species counts and/or pathogenic bacteria only at the post-operative examination, was observed in 10 patients. Six patients showed increased Candida species counts at the post-operative examination compared with the pre-operative examination. In five patients, pathogenic bacterial species were detected at the post-operative examination but not at the pre-operative examination. One patient had detectable pathogenic bacterial species only at the post-operative examination along with increased Candida species counts. Our findings suggest that pre-operative oral hygiene to remove bacterial and Candida species from patients who are scheduled for surgical procedures is important for satisfactory clinical outcomes.

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model.

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.

Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis.

Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN- and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.

Analysis of retrograde ejaculation using color Doppler ultrasonography before and after transurethral collagen injection.

Transurethral bladder neck collagen injection therapy was performed in a patient with retrograde ejaculation. The phenomenon of retrograde ejaculation and its correction after the therapy were clearly demonstrated by color Doppler ultrasonography. To our knowledge this is the first report showing successful observation of retrograde ejaculation using color Doppler ultrasonography.

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Orthotopic neobladder reconstruction in elderly bladder cancer patients.

BACKGROUND: We compared the clinical results of orthotopic neobladder reconstruction in elderly patients and those in younger patients retrospectively in order to verify whether age is a critical factor in selecting a method of urinary diversion. METHODS: Following radical cystectomy for bladder cancer, 12 patients aged 75 or older and 17 patients under 75 who underwent orthotopic neobladder reconstruction between January 1992 and May 1999 were investigated in this study. The authors TS and BS were among the surgeons who performed operations for all cases. Of the 12 elderly patients, orthotopic neobladders were constructed according to Hautmann's method in nine cases, Studer's method in one case and Reddy's method in two cases. Of the 17 younger patients, these methods were employed in 12, one and four cases, respectively. Operative procedure, early and late complications, prognosis, continence and voiding pattern were investigated in these patients. RESULTS: The follow-up periods for elderly and younger groups ranged from 21.3 to 82.7 months and from 8.8 to 94.2 months, respectively. No difference in operation time, amount of bleeding or postoperative length of hospitalization was observed between elderly and younger patients. The rates of early complications in elderly and younger patients were 41.7% and 35.3%, respectively. Late complication rates were 33.3% and 47.1%, respectively. The difference in these complication rates was not statistically significant. One of the elderly and two of the younger patients had local recurrence and metastasis postoperatively. Those three patients had died of their bladder cancer. No statistically significant difference between groups was recognized in either cause-specific survival or overall survival, nor was there such a difference in relation to micturition/continence. CONCLUSION: Based on these results, we believe that because age is not a critical factor in the selection of urinary diversion method, neobladder reconstruction following cystectomy for bladder cancer is indicated in elderly patients. As stoma management is difficult for the patients, we consider orthotopic neobladder reconstruction to be the method of choice if the patients' general physical condition allows.

Gene therapy and tissue engineering for urologic dysfunction: status and prospects.

This article reviews the recent advances in gene therapy and tissue engineering for urologic dysfunction. Although the number of gene therapy-based clinical trials has increased dramatically in the field of urologic oncology, such trials are still few within the neurourologic field. Recently, new biologic approaches employing growth factors have been utilized to treat various pathological conditions. Among them, transfer of genes such as those encoding growth factors represents a promising way to deliver therapeutic proteins to malfunctioning tissues, which leads to the improvement of organ function. Tissue engineering, which may eventually be combined with gene therapy, also offers the potential to create new functional genitourinary tissue for regeneration and replacement of tissue lost as a consequence of disease. Thus, both tissue engineering and gene therapy may hold promising new solutions in the urologic field.

Anticlastogenic activity of cacao: inhibitory effect of cacao liquor polyphenols against mitomycin C-induced DNA damage.

Oxidative DNA damage has been implicated as a factor playing a role in mutagenesis and carcinogenesis. We investigated the anticlastogenic activity of cacao: the inhibitory effect of cacao liquor polyphenols on DNA strand cleavage induced by mitomycin C (MMC) in vitro and the anticlastogenic effect of cacao liquor extract against formation of micronuclei induced by MMC in bone marrow cells and peripheral blood cells of mice. In the DNA strand cleavage test, cacao liquor polyphenols inhibited cleavage of RFI DNA. In the micronuclei test, the frequency of occurrence of micronucleated cells among bone marrow cells and peripheral blood cells were reduced significantly when cacao liquor extract was administered orally to mice 6 h before intraperitoneal injection of MMC. These findings suggest that cacao liquor polyphenols are effective in preventing DNA damage, and one of the mechanisms of action might involve scavenging of active oxygen radicals generated in reactions initiated by MMC.

Expression of amphiphysin I in Sertoli cells and its implication in spermatogenesis.

Amphiphysin I is a protein concentrated in nerve terminals and involved in the endocytosis of synaptic vesicle membrane. We show here that amphiphysin I is expressed in the rat testis, localized exclusively in the Sertoli cells. In the postnatal testicular development, expression of amphiphysin I was not evident at birth, but became significant at postnatal day 15 (P15), coinciding with the onset of spermatogenesis. The expression level of amphiphysin I increased 10-fold between P15 and P25 to reach the adult level. In adult testes reversibly damaged by ethane dimethane sulphonate administration, expression of amphiphysin I did not change following the damage, whereas the protein was transiently converted into its phosphorylated form. The increase in levels of phosphorylated amphiphysin I was closely associated with the severe histological damage to germ cells. The present findings suggest that amphiphysin I in Sertoli cells is involved in spermatogenesis, probably through endocytic processes.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (1999). III. Secular changes in susceptibility].

The bacteria (Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as having urinary tract infections (UTIs) in 9 institutions in Japan were supplied between the period of August 1999 to July 2000. Then, the susceptibilities of these bacteria to various antimicrobial agents were examined and the results were compared with those obtained between 1991 and 1998. Comparison was made by classifying strains isolated from patients into those with uncomplicated UTIs and those with complicated UTIs (including with or without indwelling catheter). About E. faecalis, increase of low sensitive strains noted in the former year showed a decreasing tendency, however, one strain each with MIC of 4 micrograms/ml to vancomycin (VCM) was detected in patients with both uncomplicated and complicated UTIs. As for S. aureus, many sensitive strains to cephems, imipenem (IPM) and VCM were noted, and each MIC50 was better than that in the former years. S. aureus strains showing low susceptibility to arbekacin (ABK) were detected in patients with complicated UTIs in this year as well as in the former year, and one strain each with MIC of 16 micrograms/ml and 32 micrograms/ml was detected. Susceptibilities of E. coli were effective to all drugs except for penicillins and minocycline (MINO). Decrease of low sensitive strains was also noted in all drugs except for quinolones. Each MIC90 of ciprofloxacin (CPFX) and sparfloxacin (SPFX) in patients with complicated UTIs against E. coli was 3 degrees classes lower than that in patients with uncomplicated UTIs. As for Klebsiella pneumoniae, decrease of low sensitive strains to cephems was noted in patients with uncomplicated UTIs in 1998. In 1999, low sensitive strains decreased also in patients with complicated UTIs, and few were detected. Susceptibilities of K. pneumoniae to quinolones were effective as compared with those in the former years with the MIC80s of 0.125 microgram/ml or below without detection of low sensitive strains. One low sensitive strain of K. pneumoniae with MIC of 8 micrograms/ml was detected for gentamicin (GM). Susceptibilities of P. aeruginosa to carbapenems were notable. The MIC90 of meropenem (MEPM) and IPM was 4 micrograms/ml each which was 2 degrees better than that in 1998. Resistant P. aeruginosa strains to other drugs except for monobactams decreased in 1999.